The aim of this project is to develop a high throughput screening (HTS) assay for the identification of pharmacological probes for an essential enzyme in trypanosome parasites, RNA editing ligase 1 (REL1). Such probes will be powerful tools to study the function of REL1 and related enzymes and to dissect the mechanism of RNA editing in trypanosomatids. Importantly, they will also serve as lead compounds for the development of new drugs against the devastating diseases caused by these organisms. The assay developed in this project will be a rapid, sensititive and robust "mix and measure" in vitro assay based on fluorescence resonance energy transfer (FRET). In the first aim, critical assay parameters will be identified and optimized and the established assay will be validated for robust and reproducible behaviour in a 96-well format. In the second aim, secondary screens and counterscreens will be developed which, in combination with the traditional RNA ligase assays already available, will be critical for validating compounds obtained in primary screens and for obtaining initial data on specificity. These assays will then be tested in a pilot screen against a small but diverse set of compounds. We anticipate that the screening and counter-screening assays developed in this project will provide a valuable platform for the identification of inhibitors with varying degrees of specificity, thus providing pharmacological probes not only for REL1 but also for polynucleotide ligases in general, a class of enzymes for which very few inhibitors have been described to date. In addition, specific REL1 inhibitors will provide leads for the development of new drugs against sleeping sickness, Chagas disease and Leishmaniases, diseases that cause a staggering amount of human suffering and for which safe, cheap and efficient treatments are currently not available. [unreadable] [unreadable] [unreadable]